The Use of Mudlac Transposons as Tools for Vital Staining to Visualize Clonal and Non-clonal Patterns of Organization in Bacterial Growth on Agar Surfaces

Shapiro, James A.

doi:10.1099/00221287-130-5-1169

Cited by 29 publications

(47 citation statements)

References 14 publications

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“…However, the predominant mode of differentiation observed in these young colonies was concentric and thus independent of cell lineages. These observations on colony microstructure relate directly to earlier work on the control of largescale aspects of colony morphology (15)(16)(17)19), because it was the general rule that macroscopic boundaries, such as sector edges and concentric changes in colony structure, were marked by microscopic boundaries between bacterial groups that differed in cell size, cell shape, and cell alignments. The results thus provided an affirmative answer to the question of whether populations of nonswarming bacteria display identifiable changing patterns of multicellular organization as colonies develop.…”

Section: Resultsmentioning

confidence: 68%

“…These methods were as described in previous publications (16)(17)(18)(19). As mentioned in the introduction, spot inoculations were used to initiate colony growth rather than stabbing, streaking, or spreading dilute suspensions.…”

Section: Methodsmentioning

confidence: 99%

“…Examination of fully developed bacterial colonies at both the macroscopic and microscopic levels has revealed a high degree of morphological and biochemical organization (1,2,12,(15)(16)(17)(18)(19). Large-scale patterns included both sectorial and concentric elements.…”

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confidence: 99%

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Organization of developing Escherichia coli colonies viewed by scanning electron microscopy

Shapiro¹

1987

J Bacteriol

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Colony growth was initiated by inoculating minimal glucose agar with 1-,ul. spots of a plasmid-free Escherichia coli culture and incubating at 32°C. Inoculations took place over a 3-day period, at the end of which the plates were fixed and dried for scanning electron microscopy. In this way, it was possible to examine the surfaces of colonies ranging in age from 0 to 68 h. Macroscopically, the colonies were organized into different concentric zones, and several morphological features could be seen to develop over this period. These included a shallow depression ring marking the site of inoculation, a deeper indentation ring whose position moved outward as the colony grew, an expanding plateau region between the two rings, a mound outside the indentation ring, and a flat brim extending onto the substrate which was either present or absent at different times. Microscopically, a variety of cell morphologies and cell arrangements were detected. Upon inoculation, the bacteria accumulated at the periphery of the inoculation spot but showed no other kind of order. (20,24). Observations such as these indicated that regulatory systems could control the behavior of groups of procaryotic cells and that it should be possible to monitor specific multicellular morphogenetic events during bacterial colony development. This idea is not unprecedented. Other authors have proposed similar ideas based on studies of colony structure and variation (1,5,12).There are a number of studies of the early stages of bacterial colony development in the literature (7, 10, 13). These studies generally involved cultures growing on special slides or chambers and were limited to the first few hours of growth when most of the individual bacteria could be distinguished. To provide information about cellular changes at later stages of colony development, the scanning electron microscope (SEM) was used to examine the surface structures of E. coli colonies growing on agar medium in normal petri dishes. Previous work had shown that the SEM could be used to provide detailed information at the cellular level about the structure of mature Pseudomonas putida colonies without gross distortion of colony organization (18). The present study extended the earlier results by examining the colonies produced by a single culture at different stages of development, thereby documenting how patterns of multicellular organization could change over time.The background for these SEM studies was an investigation of bacterial morphogenesis on agar substrates, using time-lapse photography and video recording. The time-lapse studies concerned the growth of E. coli colonies and the formation of swarm colonies by Proteus mirabilis. The results significantly influenced my descriptions and interpretations of the SEM pictures in this report and thus require a brief summary. Kinetic analysis of living E. coli colonies revealed that expansion was not a uniform process but rather that different phases of activity could be discerned. To obtain the colonies for SEM examination, pla...

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Section: Resultsmentioning

confidence: 68%

Section: Methodsmentioning

confidence: 99%

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Organization of developing Escherichia coli colonies viewed by scanning electron microscopy

Shapiro¹

1987

J Bacteriol

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“…Remarkably, such rings have been described previously when monitoring formation of araB-lacZ fusions carried by the bacteriophage Mu (46). Although these rings formed under quite different conditions and were continuous rings of ␤-galactosidase expression, rather than individual papilla, they are a result of Mu transposition, requiring transposase activity and Mu ends (48).…”

Section: Resultsmentioning

confidence: 86%

Genetic Evidence that GTP Is Required for Transposition of IS 903 and Tn 552 in Escherichia coli

et al. 2005

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Surprisingly little is known about the role of host factors in regulating transposition, despite the potentially deleterious rearrangements caused by the movement of transposons. An extensive mutant screen was therefore conducted to identify Escherichia coli host factors that regulate transposition. An E. coli mutant library was screened using a papillation assay that allows detection of IS903 transposition events by the formation of blue papillae on a colony. Several host mutants were identified that exhibited a unique papillation pattern: a predominant ring of papillae just inside the edge of the colony, implying that transposition was triggered within these cells based on their spatial location within the colony. These mutants were found to be in pur genes, whose products are involved in the purine biosynthetic pathway. The transposition ring phenotype was also observed with Tn552, but not Tn10, establishing that this was not unique to IS903 and that it was not an artifact of the assay. Further genetic analyses of purine biosynthetic mutants indicated that the ring of transposition was consistent with a GTP requirement for IS903 and Tn552 transposition. Together, our observations suggest that transposition occurs during late stages of colony growth and that transposition occurs inside the colony edge in response to both a gradient of exogenous purines across the colony and the developmental stage of the cells.Transposons are defined as distinct regions of DNA that have the ability to move from one genomic location to another, a process mediated by element-encoded transposases (8, 9). While the main focus of transposition research has been in determining the detailed mechanisms of transposition, relatively little attention has been paid to how transposition is regulated in vivo or the in vivo requirements for the process (35). Transposon movement can result in gene inactivation (by insertional inactivation or deletion) or activation (by creation or introduction of promoters upstream of genes). In addition, intramolecular transposition can result in extensive DNA rearrangements, including deletions, inversions, and duplications of chromosome segments; these events are thought to play an important role in facilitating genome evolution (7-9). As the consequences of transposition can be either dire or favorable for the host organism, it is intuitive that the host has the capability to regulate this process. There are multiple examples of how transposons regulate their own movement, but very little work has focused on the role played by the host in regulating transposition. To address this, we have generated an insertion mutant library of Escherichia coli, which we have used to screen for host genes involved in regulation of IS903 transposition (E. Twiss, A. Coros, N. Tavakoli, and K. M. Derbyshire, unpublished data).The first comprehensive genetic screen for host mutants affecting transposition was carried out in 1985; it revealed a role for dam methylation in decreasing the rate of IS10, IS903, and IS50 transposi...

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“…Although the microenvironment within bacterial colonies is poorly understood, several groups have recently shown that cell organization in them is highly ordered and that colony development is a morphogenetic process governed by genetic and environmental factors (3,5,(17)(18)(19). In Bacillus subtilis colonies, for example, cell separation or its absence following septation, as well as cell motility and chemotactic behavior or the lack thereof, are phenotypes that strongly influence colony form (10,13).…”

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confidence: 99%

Patterns of reporter gene expression in the phase diagram of Bacillus subtilis colony forms

Mendelson

Salhi

1996

J Bacteriol

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Factors governing the morphogenesis of Bacillus subtilis colonies as well as the spatial-temporal pattern of expression of a reporter gene during colony development were examined by systematically varying the initial nutrient levels and agar concentrations (wetness), the relative humidity throughout incubation, and the genotype of the inoculum. A relationship between colony form and reporter gene expression pattern was found, indicating that cells respond to local signals during colony development as well as global conditions. The most complex colony forms were produced by motile strains grown under specific conditions such that cells could swim within the colony but not swarm outward uniformly from the colony periphery. The wetness of the growth environment was found to be a critical factor. Complex colonies consisted of structures produced by growth of finger-like projections that expanded outward a finite distance before giving rise to a successive round of fingers that behaved in a similar fashion. Finger tip expansion occurred when groups of cells penetrated the peripheral boundary. Although surfactin production was found to influence similar colony forms in other B. subtilis strains, the strains used here to study reporter gene expression do not produce it. The temporal expression of a reporter gene during morphogenesis of complex colonies by motile strains such as M18 was investigated. Expression arose first in cells located at the tips of fingers that were no longer expanding. The final expression pattern obtained reflects the developmental history of the colony.

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The Use of Mudlac Transposons as Tools for Vital Staining to Visualize Clonal and Non-clonal Patterns of Organization in Bacterial Growth on Agar Surfaces

Cited by 29 publications

References 14 publications

Organization of developing Escherichia coli colonies viewed by scanning electron microscopy

Organization of developing Escherichia coli colonies viewed by scanning electron microscopy

Genetic Evidence that GTP Is Required for Transposition of IS 903 and Tn 552 in Escherichia coli

Patterns of reporter gene expression in the phase diagram of Bacillus subtilis colony forms

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